Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
V6.5 mESC
cell line
V6.5 mESC
cell type
Mouse embryonic stem cells
transgenes
combined input file
days post transgene induction
2 days
chip antibody
input

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
FLAG-tagged TFs were immunoprecipitated using FLAG M2 antibody conjugated beads (Sigma F2426). Approximately 60-100 million cells were used per replicate. Beads (~100uL slurry per sample) were washed thrice with IP dilution buffer (1% Triton X-100, 2mM EDTA pH8.0, 20mM Tris-HCl pH8.0, 150mM NaCl, 1mM DTT, 100uM PMSF), blocked overnight in IP dilution buffer with 0.1% BSA and 0.06% sheared salmon sperm DNA (Thermo Fisher; AM9680), then washed again three times with IP dilution buffer before use. Nuclei were isolated by incubating with cell lysis buffer (5mM HEPES pH7.9, 85mM KCl, 0.5% NP40, 100uM PMSF, protease inhibitors from Roche) for 10min on ice and centrifuged at 5000rpm for 5min at 4°C, then lysed with nuclear lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS, 100uM PMSF, protease inhibitors) for 10min on ice. Chromatin was sheared using either the Bioruptor (Diagenode) or Covaris sonicator until DNA was fragmented to 200-500bp. Sheared chromatin was diluted using 3X volume of IP dilution buffer and pre-cleared for at least 4hr at 4°C using IgG beads (Sigma A0919). 1% of pre-cleared chromatin was kept as input, and the remainder was incubated with FLAG beads overnight at 4°C. Beads were washed 8 times with IP wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 250mM NaCl, 1% NP40, 0.05% SDS, 100uM PMSF) and once with TE buffer with 100uM PMSF. Beads and 1% input samples were reverse cross-linked overnight in IP elution buffer (50mM NaHCO3, 1% SDS) at 65°C. Isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water. ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier's protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
180151395
Reads aligned (%)
98.3
Duplicates removed (%)
92.3
Number of peaks
7162 (qval < 1E-05)

mm9

Number of total reads
180151395
Reads aligned (%)
98.2
Duplicates removed (%)
92.4
Number of peaks
7071 (qval < 1E-05)

Base call quality data from DBCLS SRA